Method for isolating DNA from biological samples

ABSTRACT

The present invention relates to an improved process for isolating DNA from biological samples, particularly from human whole blood.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a United States national phase filing under 35U.S.C. §371 of international (PCT) application No. PCT/EP02/10823, filedSep. 26, 2002, designating the United States, which application claimspriority to German Patent Appln. No. 101 47 439.3, filed Sep. 26, 2001.

The present invention relates to an improved process for isolating DNAfrom biological samples, particularly from human whole blood.

The purification of nucleic acids plays a central role in molecularbiology. The purified DNA is used among other things as a startingmaterial for diagnosis based on nucleic acids, for establishing geneticprofiles and for pharmacogenomics. Therefore, the isolation of nucleicacids such as DNA and RNA from biological samples, such as for exampleblood, body secretions, tissue samples, urine, stools and the like, forsubsequent use in genetic analyses is of particular importance.

In the methods known from the prior art for recovering nucleic acids,DNA is isolated from cells and tissues by lysing the starting materialsunder highly denaturing and reducing conditions, partly usingprotein-degrading enzymes, purifying the nucleic acid fractions obtainedby means of phenol/chloroform extraction processes and recovering thenucleic acids from the aqueous phase by dialysis or ethanolprecipitation [Sambrook, J., Fritsch, E. F. in T. Maniatis, C S H,“Molecular Cloning”, 1989].

These long-established methods of isolating DNA from cells andparticularly from tissues have serious disadvantages, however: on theone hand they are very time-consuming and require considerableexperimental effort. In addition, such procedures are associated with anacute risk of damage to the health of the people carrying out theisolation owing to the chemicals which have to be used, such aschloroform or phenol.

The inevitable consequence of the disadvantages described above was thatover time alternative methods would be developed for isolating nucleicacids from different biological starting materials, by which thelaborious and health-damaging phenol/chloroform extraction of nucleicacids could be avoided and the time taken should be reduced.

All these proposed methods known from the prior art are based on amethod developed and first described by Vogelstein and Gillespie [Proc.Natl. Acad. Sci. USA, 1979, 76, 615-619] for the preparative andanalytical purification of DNA fragments from agarose gels. The methodcombines the dissolving of the agarose containing DNA bands in achaotropic saline solution with binding of the DNA to carrier particles.The fixed DNA is then washed with a washing solution (20 mM EDTA; 50%v/v ethanol) and then detached from the carrier particles.

Hitherto, this method has undergone a number of modifications and isstill used for different processes for extracting and purifying nucleicacids from various sources (Marko, M. A., Chipperfield, R. and Birnboim,H. G., 1982, Anal. Biochem. 121, 382-387).

In addition, worldwide, there are now a number of reagent systems,particularly for purifying DNA fragments from agarose gels and forisolating plasmid DNA from bacterial lysates, but also for isolatinglonger-chained nucleic acids (genomic DNA, total cell RNA) from blood,tissues or cell cultures.

Many of these commercially available purification systems are based onthe reasonably well known principle of binding nucleic acids to mineralcarriers in the presence of solutions of different chaotropic salts. Inthese systems, suspensions of finely ground glass powder, diatomaceousearth or silica gels are used as carrier materials.

A process for isolating nucleic acids which may be implemented for anumber of different applications is disclosed in U.S. Pat. No.5,234,809. This discloses a process for isolating nucleic acids fromstarting materials which contain nucleic acids by incubating thestarting material with a chaotropic buffer and a DNA-binding solidphase. The chaotropic buffers bring about both lysing of the startingmaterial and binding of the nucleic acids to the solid phase. Theprocess is used to isolate nucleic acids from small samples and isparticularly used in practice for isolating viral nucleic acids.

The physicochemical principle of the prior-art systems currently usedand commercially available for isolating nucleic acids based on thebinding of nucleic acids to the surfaces of mineral carriers is supposedto consist of the disruption of the higher structures of the aqueousmedium by which the nucleic acids are adsorbed on to the surface ofmineral materials, particularly glass or silica particles. Thedisruption of the higher structures of the aqueous medium is alwayseffected in the presence of chaotropic ions and is virtuallyquantitative when high concentrations of these ions are present. On thephysicochemical basis described above a number of commercially availablesystems for isolating nucleic acids contain buffer compositions withchaotropic salts having high ionic strength which are regarded asnecessary for the binding of nucleic acids to a nucleic acid-bindingsolid phase.

However, among the serious disadvantages of the process are the factthat the lysing effected by the chaotropic buffers is by no meanssuitable for all materials or is extremely inefficient for largerquantities of starting materials and is extremely time-consuming. Inaddition, different concentrations of different chaotropic buffers haveto be used for different situations, which proves very complicated.

On the other hand, the isolation of nucleic acids based on a salting outstep is known from the prior art [L. A. Salazar et al., Clin. Chem. 44(1998) 1748; S. A. Miller et al., Nucleic Acid Res., 16(3) (1988) 1215].These processes has the disadvantage, however, that during the processthe reaction vessel has to be changed at least once, with the risk ofmixing up the samples. Furthermore, the salted out proteins can only beseparated from the nucleic acid which is to be purified by using theexperimentally laborious method of centrifugation.

Another process for purifying DNA obtained from blood is disclosed by T.A. Ciulla [Analytical Biochemistry, 174 (1988) 485]. In this, first ofall the cell nuclei are liberated by a lysing step. The DNA containedtherein is isolated using guanidinium-iso-thiocyanate andβ-mercaptoethanol and subsequent precipitation with 2-propanol. However,this process includes relatively time-consuming steps for resuspensionand centrifuging. Moreover, this process uses a lysing buffer which issusceptible to microbial growth and has to be used in a nine-fold excessin relation to the blood. Furthermore, β-mercaptoethanol is classed as atoxic substance.

The aim of the present invention is therefore to provide a process whichovercomes the disadvantages of the processes known from the prior art.

In particular, the problem of the present invention is to avoid the useof toxic or corrosive substances in the isolation of DNA.

Furthermore, the present invention sets out to propose a lesstime-consuming and less laborious process for isolating DNA.

A further aim of the present invention is to provide a process which canbe carried out without changing the reaction vessel—as a so-called“one-pot reaction”—and thereby minimises the risk of confusion orcontamination of the samples.

The above problems are solved according to the invention by mixing thebiological sample with a lysing reagent in a reaction vessel. In thenext step the cell components which contain DNA are separated from theother cell components. This separation may be done by centrifuging, forexample.

The DNA contained in the sediment is then separated from otherimpurities such as proteins, for example. This separation is achieved byresuspending the sediment in a saline solution. Optionally, thecontaminants may also be separated from the DNA by heating and/or by theuse of enzymes. The DNA is subsequently precipitated by the addition ofalcohol and the precipitate is separated from the solution. This may bedone, for example, by centrifuging or by winding the DNA on a glasshook. After washing with a washing fluid in which salts dissolve but notthe DNA, followed by drying, the DNA is resuspended in a suitablebuffer.

Examples of biological samples containing nucleic acid may be cell-freesample material, plasma, body fluids, such as blood, buffy coat, cells,leukocyte fractions, crusta phlogistica, sputum, urine, sperm ororganisms (single- or multi-celled organisms; insects etc) or eukaryotesor prokaryotes.

In particular, blood (human whole blood), buffy coat, leukocytefractions and cell cultures are suitable for carrying out the processaccording to the invention.

Blood (human whole blood) is most particularly suitable for carrying outthe process according to the invention.

Suitable lysing reagents for carrying out the process according to theinvention are the detergent-containing lysing reagents known from theprior art. Large numbers of suitable detergents are known from the priorart.

Lysing reagents which are particularly suitable for solving the problemaccording to the invention are those which contain detergents selectedfrom among the groups Triton, Tween, NP-40 or mixtures of substancesfrom these groups. Among these groups, the Triton group is particularlysuitable, while Triton X-100 (octylphenol polyethoxylate) isparticularly preferred.

According to the invention, lysing reagents based on 0.5 to 7.5% byvolume of detergent are preferably used, while a content of 1.0 to 5.0%by volume is particularly preferred.

If desired, salts of mono- or divalent cations and complexing agents maybe added, individually or in combination, to the lysing reagent.

According to the invention, lysing reagents based on 0.5 to 200 mM ofmagnesium chloride, 0.5 to 200 mM potassium chloride and 0.5 to 100 mMethylenediamine tetraacetate and/orethylenedioxy-bis-(ethylenenitrilo)tetraacetic acid are preferably used.

Most preferably, lysing reagents based on 1 to 30 mM magnesium chloride,1 to 20 mM potassium chloride and 1 to 10 mM ethylenediaminetetraacetate and/or 1 to 10 mMethylenedioxy-bi-(ethylenenitrilo)tetraacetic acid are used.

If desired, one or more buffers may be added to the lysing reagent. ThepH of the lysing reagent may be varied over a wide range and in order tocarry out the process according to the invention is preferably in therange from pH 2 to 10, more preferably in the range from pH 3 to 9. Thebuffer systems known to the skilled man for adjusting the pH may beused. According to the invention, buffer systems based ontris(hydroxymethyl)aminomethane are preferably used. Most preferably, abuffer system based on 0.5 to 250 mM tris(hydroxymethyl)aminomethanehydrochloride is used.

Most preferably, a lysing buffer is used which contains 13 mMtris(hydroxymethyl)aminomethane hydrochloride, pH 8.0, 10 to 14.5 mMmagnesium (II)chloride hexahydrate, 10 mM potassium chloride, 2 to 5 mMethylenediamine tetraacetate and/or 1 to 2 mMethylenedioxy-bis(ethylenenitrilo)tetraacetic acid as well as 2% byvolume of Triton X-100.

Suitable saline solutions for resuspending the sediment containing thenucleic acid in order to carry out the process according to theinvention are the solutions known from the prior art for this purpose,based on high salt concentrations, particularly using chaotropic salts.By chaotropic salts are meant according to the invention salts whichhave a high affinity for water and therefore form a large hydrate shell;suitable chaotropic salts are known in large numbers from the prior art.

According to the invention, solutions containing urea, guanidiniumiso-thiocyanate or guanidinium hydrochloride are preferably used,individually or in combination, the latter being particularly preferred.

In particular, solutions based on 0.5 to 7.0M guanidinium hydrochlorideare preferably used to solve the problems according to the invention.

Solutions based on 2.0 to 4.0M guanidinium hydrochloride are mostpreferably used.

Denaturing solutions of this kind may optionally contain othersubstances such as complexing agents or buffers. The pH of thedenaturing buffer may vary over a wide range in order to carry out theprocess according to the invention; it preferably has a pH in the rangefrom 3 to 10 and most preferably in the range from 7.5 to 9.5. Thebuffer systems known to the skilled man may be used to adjust the pH.According to the invention, buffer systems based ontris(hydroxymethyl)aminomethane are preferably used. Most preferably, abuffer system based on 0.5 to 250 mM of tris(hydroxymethyl)aminomethanehydrochloride is used.

Most preferably, a denaturing buffer containing 3 M guanidiniumhydrochloride and 50 mM tris(hydroxymethyl)aminomethane hydrochloride,pH 8.5, is used.

In order to assist the denaturing activity of the denaturing buffer thesample may optionally be heated. The reaction temperature of thisoptional step of the process according to the invention is within therange from 15 to 95° C., preferably in the range from 30 to 85° C. andthis step is particularly preferably carried out at a temperature of 65°C.

In order to further separate the DNA from impurities the sample mayoptionally be treated with enzymes. The process according to theinvention may conveniently be carried out using proteases, lipases,amylases and other breakdown enzymes known in the art or mixturesthereof.

The DNA may expediently be precipitated in order to carry out theprocess according to the invention by means of alcohols known from theprior art such as straight-chain or branched C₁-C₄ alkanols. Propan-2-ol(iso-propanol) and ethanol have proved particularly suitable.

The washing fluid used is, in particular, a mixture of a straight-chainor branched C₁-C₄ alkanol with water, while aqueous solutions of ethanolare particularly preferred. A 70% by volume solution of ethanol in wateris particularly preferred.

Suitable “low salt solutions” for resuspending the DNA precipitatesinclude all liquids capable of dissolving DNA. Solutions or suitablebuffers are also known in large numbers from the prior art.

According to the invention, low salt solutions (solutions with a low ionstrength) are preferred, this category including water, according to theinvention.

Such solutions may optionally contain other substances such ascomplexing agents or buffers. The pH of the buffer may vary over a widerange and is preferably in the range pH 6 to 10 and more preferably inthe range pH 7.5 to 9.5. The buffer systems known to the skilled personin the art may be used to adjust the pH. According to the invention,buffers based on tris(hydroxymethyl)aminomethane are preferably used. Abuffer system based on 0.5 to 150 mM tris(hydroxymethyl)aminomethanehydrochloride is particularly preferred.

Most particularly preferred as a hydrogenation buffer is a solutioncontaining 10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 8.5.

Explanation of the Figures:

FIG. 1 shows the electrophoretic analysis of a DNA isolated from humanwhole blood according to Example 1.

FIG. 2 shows the electrophoretic analysis of a polymerase chain reaction(PCR) with the aim of amplifying a single-copy gene (human analog ofgiant larvae gene)

FIG. 3 shows the electrophoretic analysis of a DNA isolated from a cellculture according to Example 3

The Examples that follow are intended to illustrate the presentinvention:

EXAMPLE 1

DNA Isolation from Human Whole Blood

Human whole blood is pipetted into a reaction vessel containing 2.5volumes of lysing buffer (13 mM Tris-Cl; 10 mM KCl; 14.5 mM MgCl₂x6H₂O;2 mM EDTA; 2.0% Triton X-100; pH 8). After inverting 5 times, the lysateis centrifuged off, thus sedimenting DNA-containing cell components suchas cell nuclei and mitochondria. The supernatant is discarded. Then thesediment is homogenised in denaturing buffer (3 M guanidinium HCl; 50 mMTris-Cl pH 8.5) by vortexing or pipetting up and down. Proteins areeliminated by QIAGEN Protease in a 10 min incubation at 65° C. The DNAis precipitated from this solution by the addition of 1 volume ofisopropanol and centrifugation. After the supernatant has been decantedoff, the DNA pellet is washed with 70% ethanol, centrifuged again, thesupernatant is removed completely, the DNA is dried and resuspended in alow salt buffer or water.

The following Table 1 shows the results from 6 DNA purifications carriedout in parallel, in each case from 3 ml human whole blood which has beenanticoagulated with potassium-EDTA:

TABLE 1 Prep.-No. Yield [μg] A260/A280 1 62 1.84 2 76 1.92 3 67 1.94 468 1.93 5 66 1.92 6 58 1.95

The average yield of the 6 extractions is 66 μg, corresponding to 98% ofthe yield to be expected theoretically from the leukocyte cell count.The coefficient of variation is 8%.

In FIG. 1 in each case 2 μl of the DNA solutions obtained are applied toa 0.8% agarose gel.

FIG. 2 shows the electrophoretic analysis of a polymerase chain reaction(PCR) with the aim of amplifying a single copy gene (human analog ofgiant larvae gene) using purified genomic DNA as matrix. The experimentshows that all six extractions yield pure DNA which can be amplifiedwithout any problems.

EXAMPLE 2

DNA Isolation from Buffy Coat

The purification of DNA from Buffy Coat is carried out using the samemethod as described in Example 1 for whole blood. Unlike in the wholeblood procedure [sic]

Table 2 shows the results of 3×6 purifications carried out in parallelwith increasing amounts (200; 300; 500 μl) of Buffy Coat:

TABLE 2 Vol. Buffy Prep.- Yield Average Coeff. Coat No. [μg] A260/A280Yield [μg] var. [%] 200 μl 1 47 1.80 2 52 1.82 3 50 1.80 4 51 1.83 5 531.81 6 49 1.83 50 4 300 μl 7 74 1.83 8 75 1.84 9 71 1.85 10 75 1.84 1173 1.83 12 70 1.85 73 3 500 μl 13 125 1.83 14 111 1.85 15 118 1.84 16122 1.83 17 118 1.84 18 122 1.86 119 4

The experiment shows that the yield increases in linear manner inrelation to the starting volume of Buffy Coat.

EXAMPLE 3

DNA Isolation from Cell Culture Cells

The purification of DNA from cell culture cells (HeLa cell line) wascarried out using the same method as described in Example 1 for wholeblood.

Table 3 shows the results from 8 DNA purifications carried out inparallel, in each case from 1×10⁶ cells per 200 μl PBS:

TABLE 3 Prep.-No. Yield [μg] A260/A280 1 21 1.91 2 20 1.91 3 20 1.92 421 1.93 5 19 1.94 6 19 1.94 7 18 1.93 8 19 1.95

The average yield of the 8 extractions is 20 μg. The coefficient ofvariation is 6%.

In FIG. 3 in each case 15 μl of the DNA solutions obtained were appliedto a 0.8% agarose gel.

EXAMPLE 4

DNA Isolation from Human Whole Blood with Various Modifications to theProcedure

The purification of DNA was carried out as described in Example 1. Inone alternative form of the procedure no QIAGEN Protease is added. In asecond alternative form of the procedure again no QIAGEN Protease isadded and furthermore the incubation at 65° C. is omitted. All the othersteps of the 3 variants are identical.

Table 4 summarises the results of this experiment:

TABLE 4 yield hugl blood volume yield [%] CV [%] 260/280 220/260 PCR [%]+Protease/+65° C. ≦500 μl 104 3 1.84 0.79 93 ≦3 ml 93 10 1.87 0.73 98≦10 ml 94 8 1.85 0.71 98 Average 97 7 1.85 0.75 97 −Protease/+65° C.≦500 μl 94 5 1.84 0.78 98 ≦3 ml 86 12 1.86 0.75 94 ≦10 ml 84 11 1.860.75 95 Average 88 9 1.85 0.76 96 −Protease/−65° C. ≦500 μl 94 7 1.850.83 90 ≦3 ml 74 14 1.78 0.90 100 ≦10 ml 85 10 1.85 0.78 100 Average 8410 1.83 0.84 97

All three alternative methods yield DNA of comparable purity andamplifiability.

EXAMPLE 5

Use of Lysing Reagents with Various Salts:

Genomic DNA is purified from human whole blood according to Example 1.To lyse the blood, lysing buffer with different salts are used. Thefollowing Table summarises the alternative lysing buffers used with thecorresponding relative yields:

TABLE 5 Buffer Yield [%] 13 mM Tris-Cl; 10 mM KCl; 14.5 mM MgCl₂; 100 2mM EDTA; 2% Triton X-100; pH 8 20 mM Tris-Cl; 10 mM KCl; 5/10 mM MgCl₂;105/96 2 mM EDTA; 2% Triton X-100 20 mM Tris-Cl; 10 mM KCl; 14.5 mMMgCl₂; 108/103/103 10/20/50 mM (NH₄)₂SO₄; 2 mM EDTA; 2% Triton X-100 20mM Tris-Cl; 10 mM KCl; 15 mM MgCl₂; 102/93/97 150/200/250 mM (NH₄)₂SO₄;2 mM EDTA; 2% Triton X-100 20 mM Tris-Cl; 10 mM KCl; 1/2.5 mM ZnCl₂;108/88 2 mM EDTA; 2% Triton X-100 20 mM Tris-Cl; 10 mM KCl;91/94/96/100/99 1/2.5/3/4/5 mM ZnCl₂; 5 mM MgCl₂; 2 mM EDTA; 2% TritonX-100 20 mM Tris-Cl; 10 mM KCl; 15 mM CaCl₂; 111 2 mM EDTA; 2% TritonX-100 20 mM Tris-Cl; 10 mM KCl; 10 mM CaCl₂; 104 2 mM MgCl₂; 2 mM EDTA;2% Triton X-100

EXAMPLE 6

Use of Lysing Reagents with Different Buffer Substances/pH Values:

Genomic DNA is purified from human whole blood according to Example 1.To lyse the blood, lysing buffer with different buffer substances/pHvalues are used. The following Table summarises the alternative lysingbuffers used with the corresponding relative yields:

TABLE 6 buffer Yield [%] 13 mM Tris-Cl pH 8.0; 10 mM KCl; 100 14.5 mMMgCl₂; 2 mM EDTA; 2% Triton X-100 50/100 mM Na-acetate pH 4.0; 10 mMKCl; 107/110 15 mM MgCl₂; 2 mM EDTA; 2% Triton X-100 50/100 mMglycine-HCl pH 3.0; 10 mM KCl; 99/106 15 mM MgCl₂; 2 mM EDTA; 2% TritonX-100 50/100/160 mM lysine-HCl pH 3.0/3.3/3.4; 103/107/124 10 mM KCl; 15mM MgCl₂; 4 mM EGTA; 2% Triton X-100 50/100/160 mM lysine-HCl pH3.3/3.5/3.7; 109/101/100 10 mM KCl; 15 mM MgCl₂; 2 mM EGTA; 2% TritonX-100 50/100/160 mM lysine-HCl pH 4.3/4.7/4.6; 109/116/113 10 mM KCl; 15mM MgCl₂; 2% Triton X-100 50 mM ornithine-HCl pH 3.0; 10 mM KCl; 11114.5 mM MgCl₂; 2 mM EGTA; 2% Triton X-100

EXAMPLE 7

Use of Lysing Reagents with Different Complexing Agents:

Genomic DNA is purified from human whole blood according to Example 1.To lyse the blood, lysing buffers are used with different complexingagents. The following Table summarises the alternative lysing buffersused with the corresponding relative yields(EGTA=ethylenedioxy-bis-(ethylenenitrilo)-tetra-acetic acid;DTPA=diethylenetriamine-penta-acetic acid):

TABLE 7 Buffer Yield [%] 13 mM Tris-Cl pH 8.0; 10 mM KCl; 100 14.5 mMMgCl₂; 2 mM EDTA; 2% Triton X-100 13 mM Tris-Cl pH 8.0; 10 mM KCl;97/113/101/103 14.5 mM MgCl₂; 3/4/4.5/5 mM EDTA; 2% Triton X-100 13 mMTris-Cl pH 8.0; 10 mM KCl; 124 14.5 mM MgCl₂; 1 mM EGTA; 2% Triton X-10013 mM Tris-Cl pH 8.0; 10 mM KCl; 106 14.5 mM MgCl₂; 2 mM EDTA; 1 mMEGTA; 2% Triton X-100 13 mM Tris-Cl pH 8.0; 10 mM KCl; 101/99 14.5 mMMgCl₂; 1/2 mM DTPA; 2% Triton X-100 13 mM Tris-Cl pH 8.0; 10 mM KCl;95/102 14.5 mM MgCl₂; 1/2 mM DTPA; 2 mM EDTA; 2% Triton X-100 13 mMTris-Cl pH 8.0; 10 mM KCl; 79/197 14.5 mM MgCl₂; 1/2 mM Na₅P₃O₁₀; 2%Triton X-100 13 mM Tris-Cl pH 8.0; 10 mM KCl; 95/92 14.5 mM MgCl₂; 1/2mM Na₅P₃O₁₀; 2 mM EDTA; 2% Triton X-100 20 mM Tris-Cl pH 8.0; 10 mM KCl;110/102 15 mM MgCl₂; 1/2 mM EGTA; 1% Triton X-100 20 mM Tris-Cl pH 8.0;10 mM KCl; 111/140 15 mM MgCl₂; 2/4 mM EGTA; 2% Triton X-100

EXAMPLE 8

Use of Lysing Reagents with Different Detergent Concentrations:

Genomic DNA is purified from human whole blood according to Example 1.To lyse the blood, lysing buffers are used with different detergentconcentrations. The following Table summarises the alternative lysingbuffers used with the corresponding relative yields:

TABLE 8 Buffer Yield [%] 13 mM Tris-Cl pH 8.0; 10 mM KCl;103/100/103/102/98 14.5 mM MgCl₂; 2 mM EDTA; 1/2/2.5/3/4% Triton X-100

EXAMPLE 9

Use of Saline Solutions for Resuspending the Material Containing DNAwith Different Buffer Substances/pH Values:

Genomic DNA is purified from human whole blood according to Example 1.The lysing reagent used is 13 mM Tris-Cl pH 8.0; 10 mM KCl; 14.5 mMMgCl₂; 1 mM EGTA; 2% Triton X-100. To resuspend the material containingDNA saline solutions are used with different buffer substances/pHvalues. The following Table summarises the alternatives used with thecorresponding relative yields:

TABLE 9 Buffer Yield [%] 3 M guanidinium hydrochloride; 50 mM Tris-Cl100 pH 8.5 3 M guanidinium hydrochloride; 5 mM CaCl₂; 118 50 mM Tris-ClpH 8.5 3 M guanidinium hydrochloride; 5 mM CaCl₂; 129 50 mM Tris pH 9.53 M guanidinium hydrochloride; 50 mM glycine-NaOH 124 pH 10.0 3 Mguanidinium hydrochloride; 5 mM CaCl₂; 124 50 mM glycine-NaOH pH 10.0

EXAMPLE 10

Use of Saline Solutions for Resuspending the Material Containing DNAwith Different Chaotropic Salts:

Genomic DNA is purified from human whole blood according to Example 1.The lysing reagent used is 13 mM Tris-Cl pH 8.0; 10 mM KCl; 14.5 mMMgCl₂; 1 mM EGTA; 2% Triton X-100. To resuspend the material containingDNA saline solutions are used with different chaotropes. The followingTable summarises the alternatives used with the corresponding relativeyields:

TABLE 10 Buffer Yield [%] 3 M guanidinium hydrochloride; 50 mM Tris-Cl100 pH 8.5 3 M guanidinium hydrochloride; 0.1 M urea; 100 50 mM Tris-ClpH 8.5 2.5 M guanidinium hydrochloride; 0.5 M urea; 98 50 mM Tris-Cl pH8.5 2 M guanidinium hydrochloride; 1 M urea; 94 50 mM Tris-Cl pH 8.5 1.5M guanidinium hydrochloride; 1.5 M urea; 101 50 mM Tris-Cl pH 8.5 1.5 Murea; 50 mM Tris-Cl pH 8.5 86

EXAMPLE 11

Different Temperatures During the Protease Incubation

Genomic DNA is purified from human whole blood according to Example 1.The lysing reagent used is 13 mM Tris-Cl pH 8.0; 10 mM KCl; 14.5 mMMgCl₂; 1 mM EGTA; 2% Triton X-100. The incubation with protease wascarried out for 5 or 10 min at different temperatures. The followingTable summarises the results:

TABLE 11 Incubation Yield [%] 5 min 65° C. 100 10 min 65° C. 99 5 min75° C. 95 10 min 75° C. 85 5 min 85° C. 84 10 min 85° C. 70

EXAMPLE 12

Lipase Incubation

Genomic DNA is purified from human whole blood according to Example 1.The lysing reagent used is a) 13 mM Tris-Cl pH 8.0; 10 mM KCl; 14.5 mMMgCl₂; 2 mM EDTA; 1 mM EGTA; 2% Triton X-100 or b) 13 mM Tris-Cl pH 8.0;10 mM KCl; 14.5 mM MgCl₂; 2 mM EDTA; 2% Triton X-100. Variants with andwithout lipase incubation (5 min at 60° C.) before the proteaseincubation are compared:

TABLE 12 Incubation Yield [%] a) −Lipase 100 a) +Lipase 93 b) −Lipase100 b) +Lipase 91

1. A process for isolating DNA from a biological sample, comprising (a)mixing a biological sample with a lysing reagent, (b) separating theDNA-containing cell components from the other cell components in themixture, (c) resuspending the DNA-containing cell components in a salinesolution which contains a chaotropic salt, (d) removing contaminatingingredients from the resulting suspension by chemical, thermal and/orenzymatic treatment, (e) precipitating the DNA and retaining theprecipitate, (f) washing the DNA precipitate, (g) retaining and dryingthe washed DNA, and (h) optionally resuspending the DNA in a low saltsolution, wherein the isolation process is carried out in one reactionvessel and wherein the lysing reagent comprises, at least one salt of amono- or divalent cation and one or more complexing agents; wherein thelysing reagent is not susceptible to microbial growth.
 2. The processaccording to claim 1, wherein a detergent-containing lysing reagent isused as the lysing reagent.
 3. The process according to claim 2, whereinthe detergent is selected from the group consisting of Triton, Tween, orNP-40, or mixtures thereof.
 4. The process according to claim 2, whereinthe detergent is Triton X-100.
 5. The process according to claim 2,wherein the lysing reagent contains 0.5 to 7.5% by volume of detergent.6. The process according to claim 1, wherein the lysing reagent mayadditionally contain one or more buffer substances.
 7. The processaccording to claim 6, wherein the buffer substance is selected from thegroup consisting of tris(hydroxymethyl)aminomethane-hydrochloride,sodium acetate, glycine-hydrochloride, lysine-hydrochloride andornithine-hydrochloride.
 8. The process according to claim 6, whereinthe pH of the lysing reagent is in the range from 2 to
 10. 9. Theprocess according to claim 6, wherein the lysing reagent contains 13 mMTris(hydroxymethyl)aminomethane hydrochloride, pH 8.0, 10 to 14.5 mMmagnesium(II)chloride hexahydrate, 10 mM potassium chloride, 2 to 5 mMethylenediaminetetraacetate and/or 1 to 2 mMethylenedioxy-bis(ethylenenitrilo)tetra-acetic acid and 2% by volume ofTriton X-100 in aqueous solution.
 10. The process according to claim 1,wherein the saline solution for resuspending the DNA-containing materialcontains at least guanidinium hydrochloride as a chaotropic salt and mayoptionally contain urea and/or guanidinium-iso-thiocyanate as chaotropicsalts.
 11. The process according to claim 1, wherein the saline solutionfor resuspending the DNA-containing material is an aqueous, 0.5 to 7.0 Mguanidinium hydrochloride solution, which may optionally contain asfurther auxiliaries, buffer substances, detergents or complexing agents.12. The process according to claim 11, wherein the saline solution forresuspending the DNA-containing material is an aqueous, 2.0 to 4.0 Mguanidinium hydrochloride solution, which may optionally contain furtherauxiliaries such as buffer substances, detergents or complexing agents.13. The process according to claim 11, wherein the saline solutioncontains a buffer system based on tris(hydroxymethyl)aminomethane orglycine-sodium hydroxide.
 14. The process according to claim 11, whereinthe pH of the buffered saline solution is in a range from 3 to
 10. 15.The process according to claim 11, wherein the saline solution forresuspending the DNA-containing material comprises an aqueous 3 Mguanidinium hydrochloride solution and 50 mMTris(hydroxymethyl)aminomethane hydrochloride, pH 8.5.
 16. The processaccording to claim 1, wherein the removing contaminating ingredientscomprises heating the sample to a temperature in a range from 15° C. to95° C.
 17. The process according to claim 1, wherein the removingcontaminating ingredients comprises treating with enzymes to break downcontaminants.
 18. The process according to claim 1, wherein the nucleicacid is precipitated by the addition of a branched or unbranchedC₁-C₄-alkanol, or mixtures of these alcohols.
 19. The process accordingto claim 1, wherein the washing in step (f) is performed by a solutioncomprising a mixture of a branched or unbranched C₁-C₄-alkanol andwater.
 20. The process according to claim 1 wherein the low saltsolution comprises a solvent or a saline solution in which the DNAdissolves.
 21. The process according to claim 20, wherein water is usedas solvent.
 22. The process according to claim 20, wherein the salinesolution or the solvent contains a buffer based ontris(hydroxymethyl)aminomethane and/or optionally complexing agents. 23.The process according to claim 22, wherein the pH of the low saltsolution is in the range from 6 to
 10. 24. The process according toclaim 23, wherein an aqueous solution of 10 mMtris(hydroxymethyl)aminomethane-hydrochloride with a pH of 8.5 is usedas the low salt solution.
 25. The process according to claim 1, whereinhuman whole blood is used as the biological sample.
 26. The processaccording to claim 1, wherein buffy coat is used as the biologicalsample.
 27. The process according to claim 1, wherein a cell culture isused as the biological sample.
 28. A kit for performing the process ofclaim 1, said kit comprising the following aqueous solutions: (a) adetergent; (b) at least one salt of a mono- or divalent cation and oneor more complexing agents; (c) optionally a buffer system comprisingtris(hydroxymethyl)aminomethane-hydrochloride, sodium acetate,glycine-hydrochloride, lysine-hydrochloride or ornithine-hydrochloride;(d) a salt solution for resuspending the DNA-containing material saidsolution comprising chaotropic salts comprising at least guanidiniumhydrochloride which may optionally be used in admixture with urea and/orguanidinium-iso-thiocyanate; (e) enzymes for breaking down impurities;(f) a branched or unbranched C₁-C₄ alcohol, or mixtures of thesealcohols for precipitating the nucleic acids; (g) optionally a washingliquid consisting of a mixture of a branched or unbranched C₁-C₄-alkanoland water; (h) a low salt solution or a solvent in which the nucleicacids dissolve, as well as optionally buffers based ontris(hydroxymethyl)aminomethane and/or optionally complexing agents; and(i) optionally other auxiliaries for carrying out the process accordingto claim
 1. 29. The process according to claim 2, wherein the lysingreagent comprises 1.0% to 5.0% by volume of detergent.
 30. The processaccording to claim 1, wherein the at least one salt of a mono- ordivalent cation comprises 1 mM to 30 mM magnesium chloride and/or 1 mMto 20 mM potassium chloride and the complexing agent comprises 1 mM to10 mM ethylenediaminetetraacetate and/or 1 mM to 10 mMethylenedioxy-bis(ethylenenitrilo)tetra-acetic acid.
 31. The processaccording to claim 7, wherein the buffer substance is from about 0.5 mMto about 250 mM.
 32. The process according to claim 6, wherein thelysing reagent is from pH 3to pH
 9. 33. The process according to claim13, wherein the buffer system comprises 0.5 mM to 250 mMtris(hydroxymethyl)aminomethane hydrochloride.
 34. The process accordingto claim 11, wherein the saline solution is from pH 7.5 to pH 9.5. 35.The process according to claim 1, wherein the removing contaminatingingredients comprises heating to about 30° C. to about 85° C.
 36. Theprocess according to claim 17, wherein said enzymes for breaking downcontaminants are selected from the group consisting of proteases,lipases, amylases, or mixtures thereof.
 37. The process according toclaim 18, wherein the nucleic acid is precipitated by the addition ofpropan-2-ol(iso-propanol).
 38. The process according to claim 19,wherein said mixture is comprised of a solution of ethanol in water. 39.The process according to claim 38, wherein said mixture is comprised of70% by volume ethanol in water.
 40. The process according to claim 22,wherein said buffer is 150 mM tris(hydroxymethyl)aminomethanehydrochloride.
 41. The process according to claim 22, wherein the lowsalt buffer is from about pH 7.5 to about 9.5.
 42. The kit according toclaim 28, wherein said at least one salt of a mono- or divalent cationcomprises 0.5 mM to 200 mM magnesium(II)chloride and/or 0.5 mM to 200 mMpotassium chloride and said complexing agent comprises 0.5 mM to 100 mMethylenediamine tetraacetate and/or 0.5 mM to 100 mMethylenedioxy-bis(ethylenenitrilo)tetraacetic acid.
 43. The kitaccording to claim 28, wherein said at least one salt of a mono- ordivalent cation comprises 1 mM to 30 mM magnesium chloride and/or 1 mMto 20 mM potassium chloride and said complexing agent comprises 1 mM to10 mM ethylenediamine tetraacetate and/or 1 mM to 10 mMethylenedioxy-bis-(ethylenenitrilo)-tetra-acetic acid.
 44. The kitaccording to claim 28, wherein said buffer system is from about 0.5 mMto 250 mM.
 45. The kit according to claim 28, wherein said enzymes forbreaking down contaminants are selected from the group consisting ofproteases, lipases, amylases, or mixtures thereof.
 46. The kit accordingto claim 28, wherein said C₁-C₄ alcohol is propan-2-ol(iso-propanol).47. The kit according to claim 28, wherein said washing liquid iscomprised of a solution of ethanol in water.
 48. The kit according toclaim 47, wherein said solution is comprised of 70% ethanol by volume inwater.
 49. The kit according to claim 28, wherein said buffer is 150 mMtris(hydroxymethyl)aminomethane hydrochloride.
 50. The process accordingto claim 1, wherein the at least one salt of a mono- or divalent cationcomprises 0.5 mM to 200 mM magnesium chloride and/or 0.5 mM to 200 mMpotassium chloride and said complexing agent comprises 0.5 mM to 100 mMethylenediamine tetraacetate.
 51. The process according to claim 1wherein step (d) does not involve centrifugation.
 52. The processaccording to claim 1 wherein step (d) involves thermal treatment. 53.The process according to claim 1 wherein step (d) involves chemicaltreatment.
 54. The process according to claim 1 wherein step (d)involves enzymatic treatment.